This invention relates to vaccines, their preparation and use in medical treatments. More particularly, it relates to treatments for alleviating autoimmune diseases and their symptoms, to a vaccine useful therein, and to processes for preparing and using such a vaccine. In one particularly preferred aspect, the present invention relates to methods for treating and preventing atherosclerosis.
Autoimmune (immune-mediated) diseases include rheumatoid arthritis, graft versus host disease, systemic lupus erythromatosis (SLE), scleroderma, multiple sclerosis, diabetes, organ rejection, inflammatory bowel disease, psoriasis, and other afflictions. It is becoming increasingly apparent that many vascular disorders, including atherosclerotic forms of such disorders, have an autoimmune component, and a number of patients with vascular disease have circulating auto antibodies. Autoimmune diseases may be divided into two general types, namely systemic autoimmune diseases (exemplified by arthritis, lupus and scleroderma), and organ-specific (exemplified by multiple sclerosis, diabetes and atherosclerosis, in which latter case the vasculature is regarded as a specific organ).
In general terms, a normally functioning immune system distinguishes between the antigens of foreign invading organisms (non-self) and tissues native to its own body (self), so as to provide a defence against foreign organisms. Central to the proper functioning of the immune system, therefore, is the ability of the system to discriminate between self and non-self. When a patient""s immune system fails to discriminate between self and non-self and starts to react against self antigens, then an autoimmune disorder may arise.
The causes responsible for the reaction of an affected person""s immune system against self are not fully understood, and several different theories have been put forward. The immune response to an antigen is triggered by the interaction of the antigen with receptors of predetermined specificity on certain lymphocytes. It is believed that, at an early stage in development of the immune system, those lymphocytes with receptors recognizing self antigens are recognized and eliminated from the body""s system by a process of deletion. Alternatively, or in addition, such self-reactive lymphocytes may be controlled by the suppression of their activities. Both mechanisms probably occur.
The immune system of normal healthy individuals is able to identify and to react against a family of proteins which are highly conserved in nature (i.e. they have a similar structure throughout all living organisms). This family of proteins is called the stress or heat-shock proteins (HSP), and they are grouped according to their approximate molecular weights. Members of the HSP family include the HSP60 group, including, among others, proteins in the molecular weight range 50 to 100 kilodaltons. Increased production of HSP""s was first identified as a response to heat stress, but this now appears to be part of a general response to a variety of cell stresses. HSPs are normally located within cells, and their function appears to be stabilization of the structure of various proteins in stressed cells, so as to protect the cell from the protein denaturing effects of various stressors. However, it is likely that HSPs have a number of other functions which are, as yet, not fully understood. Heat shock proteins, HSP""s are discussed in some detail by William J. Welch, in an article in xe2x80x9cScientific Americanxe2x80x9d, May, 1993, page 56.
One group of the family of HSP""s, the HSP60 group, contains proteins which show about 50% identity between bacterial cells and human cells. Infections with bacteria containing HSP 65 results in an immune response in healthy humans against the bacterial HSP65, evidenced by the production of anti-HSP65 antibodies. Thus, a healthy immune system appears to be able to identify and react against self-like antigens.
In certain pathologies, for example many autoimmune diseases such as rheumatoid arthritis and scleroderma, patients also show the presence of antibodies to HSP65. In the past, this has led to conclusions that autoimmune diseases result from bacterial infection. Now it seems likely that autoimmune diseases are associated with an inappropriate control of autoimmune response. In other words, it is possible that the antibodies to HSP65 result from an autoimmune reaction initiated by HSPs from the body itself, but one which has been improperly controlled. In such cases, therefore, it should be possible to control an inappropriate autoimmune response, by stimulating the body""s natural immune control mechanisms, using a particularly and specific method of vaccination.
To stimulate the body""s immune response, a vaccine is required which will, upon injection into the host body, enable the host immune system to present the antigens contained in the vaccine to cells of the host immune system. Antigen presentation is performed by antigen presenting cells.
A vaccine to treat autoimmune diseases should contain antigens or fragments thereof (peptides) that will activate the body""s immune control mechanisms present. In addition, the antigens (peptides) should be present in a form which can be recognized by the host immune system when the vaccine is introduced into the host. Certain of the antigens may be present on intact cells. The objective of such a vaccination is to activate regulatory immune pathways, particularly those controlling autoimmune responses, thereby downregulating the autoimmune response.
The particular antigens which will activate the control mechanisms of a mammalian autoimmune system are not fully understood. It is however recognized that they may include antigens derived from lymphocyte receptors, which may function to stimulate control mechanisms, to inhibit those lymphocytes which cause pathological autoimmune responses in the patient. They may also include HSPs, such as the HSP60 group of proteins, and leucocyte surface molecules such as those of the Major Histocompatibility Complex (MHC) including MHC Class II molecules. MHC Class II molecules function physiologically to present peptides to CD4+T-cells as part of the immune response.
It is important that the lymphocyte receptors and other cell-derived molecules for vaccination of an autoimmune suffering patient be derived from cells obtained from the same patient, since this system will contain the autoimmune specificity. Receptors on other leucocytes in the blood may alternatively or additionally be important in a proposed vaccination process. The use of such a system as the basis of a vaccine may be considered analogous to the use of a particular viral antigen as a vaccine to treat and prevent disease caused by that virus. A vaccine for treating an autoimmune disease should, therefore, be prepared from a sample of the patient""s own blood. Such a vaccine may be described as an autovaccine.
For antigens to be effective in stimulating (or inhibiting) the immune system, the antigens should be presented to immune cells of the host system by antigen-presenting cells, which are naturally present in the body. Many of the antigen-presenting cells are phagocytes, which attach to the antigens, engulf them by phagocytosis, and break them down or process them. The preparation of such an autovaccine should include a process whereby the lymphocytes and other leucocytes in the vaccine, which may be a source of antigens, are modified into a form whereby they are likely to be phagocytosed by phagocytic antigen-presenting cells upon re-injection into the patient, so that the antigens or effective residues thereof are presented on the surface of an antigen-presenting cell. Then they can effect a controlling mechanism on the immune system, either inhibitory or stimulatory.
During the normal growth period of a mammalian body, tissues become reshaped with areas of cells being removed. This is accomplished by the cells"" undergoing a process called programmed cell death or apoptosis, the apoptotic cells being phagocytosed while not becoming disrupted sufficient to expose self-antigens to the immune system.
U.S. Pat. No. 3,715,430 Ryan relates to a method and apparatus for producing substantially pure oxygen having a controlled content of ozone and higher oxygen polymers. The purified oxygen gas is exposed to ultraviolet light in a wavelength of 2485 to 2537 angstrom units in order to produce 5 to 500 parts per million of ozone and higher oxygen polymers in the gas mixture. Ryan indicates that the gas produced in this manner is non-irritating to the human body and may be intravenously injected into the blood stream for therapeutic use.
U.S. Pat. No. 4,632,980 Zee et al. discloses a method of freeing blood and blood components of enveloped viruses by contacting the blood or blood product in an aqueous medium with an enveloped virus inactivating amount of ozone. The treatment is carried out at a temperature of 4xc2x0 to 37xc2x0 C., and an ozone concentration of 1-100 ppm.
U.S. Pat. No. 4,831,268 Fisch et al. provides a method for the radiation of blood to prevent arteriosclerosis related heart and vascular diseases caused by disturbances in the fat exchange. The disclosed process involves irradiating the blood in a blood conducting tube with radiation having an intensity of from about 1 mWcmxe2x88x922 to 10 mWcmxe2x88x922 in a wavelength range of from about 300 to 600 nm.
U.S. Pat. No. 4,968,483 Mxc3xcller et al. describes an apparatus for oxygenating blood, by treating an aliquot of a patient""s blood, extracorporeally, with an oxygen/ozone mixture and ultraviolet light, at a controlled temperature. The apparatus is proposed for use in haematological oxidation therapy.
U.S. Pat. No. 5,052,382 Wainwright discloses an apparatus for the controlled generation and administration of ozone. The apparatus includes a generator for generating ozone, a monitor for monitoring the ozone production, a dosage device for providing a predetermined amount of ozone administration, and a computer control device for controlling the operation of the apparatus. The patent further discloses that administration of ozone to patients is known for the treatment of viral and bacterial infections, as well as for the treatment of external sores and wounds.
It is an object of the present invention to provide a novel autovaccine useful in the prevention and alleviation of symptoms of atherosclerosis.
It is a further object of the present invention to provide a novel process for the preparation of such an autovaccine.
It is a further and more specific object of the present invention to provide a novel method for prevention and alleviation of the symptoms of atherosclerosis in a mammalian subject, preferably a human subject.
Accordingly, the present invention provides, from a first aspect, an autovaccine for prevention and treatment of atherosclerosis in a mammalian subject, and derived from an aliquot of the subject""s own blood. The autovaccine is characterized by the presence therein, in comparison with the normal blood of the patient, of at least one of the following characterizing features:
increasing numbers of lymphocytes and other leucocytes, exhibiting a condensed apoptotic-like morphology;
a release of specific proteins from the cell surface of the blood leucocytes, including the MHC Class II molecule HLA-DR, resulting in a reduction in the number of cells expressing such surface proteins;
an upregulation in the expression of certain cell surface markers for example CD-11a, a component of the ligand for the cell adhesion molecule ICAM-1; and certain T-cell regulatory molecules;
an increase in the amount of heat shock protein HSP-60 in the plasma;
a decrease in HSP-72 within the lymphocytes.
By inducing an apoptotic-like state in the lymphocytes and other leucocytes in the blood comprising the autovaccine, as evidenced by the increased numbers of lymphocytes and other leucocytes exhibiting a condensed apoptotic-like morphology therein, these cells may become more readily phagocytosed upon re-injection into the host body.
There are a number of different phagocytic cell types present in the mammalian body, including various antigen presenting cells and neutrophils. In order to facilitate phagocytosis by antigen presenting cells rather than by other phagocytes, the lymphocytes and other leucocytes present in the autovaccine of the invention are treated so that they may interact preferentially with antigen presenting phagocytic cells. Cells adhere to each other by a number of mechanisms including the expression of cell adhesion molecules. Cell adhesion molecules present on one cell type interact with specific ligand for particular adhesion molecules present on the adhering cell type. The present invention may result in a preferential interaction of cells in the autovaccine to antigen presenting cells in the host body, by upregulation, on the surface of the cells in the autovaccine, of the expression of the ligand for adhesion molecules found on antigen-presenting cells in the host body. Antigen-presenting cells express a number of cell adhesion molecules, including ICAM-1, a component of the ligand of which is CD-11a. One way by which the process of the invention may change the preferential phagocytosis of apoptosing cells is by upregulation of CD-11a.
The preparation of the autovaccine according to the present invention comprises extracting from the subject an aliquot of blood of volume about 0.01 ml to about 400 ml, and contacting the aliquot of blood, extracorporeally, with an immune system-stimulating effective amount of ozone gas and ultraviolet radiation.
The method for preventing and alleviating the symptoms of atherosclerosis in a human subject, in accordance with the present invention, comprises extracting from the patient an aliquot of blood of volume about 0.001 ml to about 400 ml, contacting the aliquot of blood, extracorporeally, with an immune system-stimulating amount of ozone gas and ultraviolet radiation, followed by administering the treated blood aliquot to the subject.
In another aspect, the present invention provides a method of treating or preventing atherosclerosis in a mammalian subject, comprising: (a) extracting an aliquot of blood from the subject; (b) treating the aliquot of blood ex vivo with at least one stressor selected from the group consisting of an oxidizing agent, ultraviolet radiation and elevated temperature; and (c) administering the aliquot of blood treated in step (b) to the subject.